Humoral and cellular response in convalescent COVID-19 lupus patients

In SLE, underlying immune dysregulation and immunosuppression may increase the susceptibility to COVID-19 and impair the humoral and adaptive response. We aimed to characterize COVID-19 infection, identifying susceptibility and severity risk factors, assessing the presence of SARS-CoV-2 IgG antibodies and analyzing the cellular response. We established a prospective cohort of lupus patients to estimate the COVID-19 incidence compared to the reference general population. Data were collected via telephone interviews and medical record review. SARS-CoV-2 IgG antibodies were measured cross-sectionally as part of routine surveillance. Longitudinal changes in antibody titers and immunological profile from convalescent COVID-19 patients were evaluated at 6, 12 and 24 week after symptom onset. From immunological studies, PBMCs from convalescent patients were extracted and analyzed by flow cytometry and gene expression analysis. We included 725 patients, identifying 29 with PCR-confirmed COVID-19 infection and 16 with COVID-19-like symptoms without PCR-testing. Of the 29 confirmed cases, 7 had severe disease, 8 required hospital admission (27.6%), 4 intensive care, and 1 died. COVID-19 accumulated incidence was higher in lupus patients. Health care workers and anti-SSA/Ro52 antibody positivity were risk factors for COVID-19 susceptibility, and hypocomplementemia for severity. SARS-CoV-2 IgG antibodies were detected in 8.33% of patients. Three fourths of confirmed COVID-19 cases developed antibodies. High prednisone doses were associated with lack of antibody response. Antibody titers declined over time (39%). Convalescent patients at week 12 after symptom onset displayed a CD8+T cell reduction and predominant Th17 with a mild Th2 response, more pronounced in severe COVID-19 disease. Longitudinal immune response analysis showed a progressive sustained increase in CD8+ T and B memory cells with a decrease of Th17 signaling. Lupus patients are at higher risk of COVID-19 infection and new susceptibility and severity risk factors were identified. Lupus patients were able to mount humoral and cellular responses despite immunosuppressive therapy.


Statistical analysis.
Descriptive statistics were used for the analysis of continuous variables expressed as mean (SD) and median with interquartile range (IQR), or range, when appropriate. Categorical variables were expressed as counts and proportion of patients (%). We compared continuous variables using t-test and ANOVA or Mann-Whitney and Kruskal-Wallis tests when appropriate, and categorial variables with Chi-squared or Fisher exact test. Multivariable logistic regression analyses were performed to control for potential confounding factors.
Standardized incidence rates and morbidity ratios were estimated using incidence data with all contemporaneous confirmed Covid-19 cases in Catalonia according to age and sex distribution. Data for Catalonian general population were obtained from the Conselleria de Salut, Generalitat de Catalunya 23 . For incidences and relative ratio results 95% confidence intervals were calculated. Longitudinal analyses were carried out using linear mixed models to deal with repeated measures. An exponential temporal covariance structure was used to account for the uneven time points. This approach allows for estimating differences between groups in each time point, as well as differences in change ("differences-in-differences") and the interaction between time and groups.
The statistical significance level was set at p < 0.05. All analyses were conducted using SAS 9.4 (SAS Institute Inc., Cary, NC, USA) statistical software.
Ethics approval and consent to participate. The study was approved by the ethics committee of Vall Hebron Institute Research (CIVIDSLE1).

Results
Patient population and COVID-19 incidence. Overall, 758 patients were contacted, of whom 725 were enrolled between March 15 and April 1, 2020. Of those, we identified 29 with confirmed RT-PCR COVID-19, 16 with COVID-19-like symptoms who did not undergo confirmatory testing, and 680 without symptoms who were not tested (Table 1 and Table S3). The overall mean follow-up period was 5.90 months (range 0.07-6.08). Patients completed an average of 5 follow-up questionnaires 3-6 throughout the study. Comparing the study groups there were more healthcare workers (HCWs) and patients with anti-SSA/Ro52 antibody positivity in the COVID-19 group. Number of patients receiving HCQ, treatment duration and serum HCQ concentration levels were similar between study groups (Table 1 and Figure S1).
Clinical characteristics and outcomes of RT-PCR confirmed COVID-19 patients. Patient Table 2). Eight patients (27.59%) required hospital admission, of whom 5 developed acute respiratory distress syndrome, 4 were admitted into ICU and one died. The accumulated incidence of ICU admission in the general population was 0.86%. Intensive care admissions were higher in lupus patients (4 out 29, 13.79%, p < 0.001). The median duration of ICU stay was 8 days (IQR 8-13 days). All patients with severe COVID-19 disease had SLE. Severe COVID-19 cases developed more bilateral ground-glass pneumonia and had higher serum ferritin and IL-6 levels ( Table 2). However, the strongest association with severe COVID-19 was hypocomplementemia (RR = 6.40, 95% CI 2.04-20.12, P = 0.007).
After 6 months follow-up, three patients with pneumonia (27.3%) had not recovered the pulmonary opacities, but lung function test progressively improved. Three thrombotic events were recorded (10.34%) including two pulmonary embolisms, and one mesenteric thrombosis. Median duration to thrombotic event post discharge was 73 days (interquartile range, 12-150). All received prophylactic low-molecular-weight heparin up to 15 days post-discharge. None had antiphospholipid antibodies.

Seroprevalence of anti-SARS-CoV-2 antibodies.
After confinement we performed a cross-sectional serological evaluation to detect past infection and assess individual antiviral humoral responses. The first measurement was obtained at a mean of 6 weeks after the onset of symptoms (range, 5 to 8), and the last at 24 weeks (range, 21 to 25). Twenty-five (8.33%) of the 300 patients evaluated had a positive SARS-CoV-2 IgG antibody test. Antibodies were found by week 6 in 21 of the 28 (75%, Fig. 3A) available patients with RT-PCR confirmed COVID-19 and in 2 patients of the 16 (12.5%) who had COVID-19-related symptoms without RT-PCR testing. The remaining 2 patients who developed an antibody response were asymptomatic (2 out of 256, 0.78%). To identify factors associated with the antibody response, we compared SARS-CoV-2 antibody-positive and nega-  www.nature.com/scientificreports/ Longitudinal serial testing of patients with RT-PCR confirmed COVID-19 (n = 28) showed a reduction in the antibody titers (Fig. 3A). Seven patients had not presence SARS-CoV-2 IgG positivity at week 6 and four lost their positivity at week 24. The patients who lost the positivity for SARS-CoV-2 IgG titers were patients receiving immunosuppressive therapy (50% with MMF). This reduction was more pronounced in patients with mild COVID-19 disease (Fig. 3B). Clinically, they had more articular and hematological manifestations (75% of them). Immunologically, no significantly changes were found in T cells, B cells or plasma cells / plasmablasts.

Longitudinal immune dynamics of COVID-19 convalescent patients. Twelve convalescent
patients' cellular immune responses were studied longitudinally (Table S6). A transient increase in the percentage of plasma cells / plasmablasts and a reduction of naïve B cells (83.3 vs 33.9%, p < 0.001) were apparent at 12 weeks (p < 0.001) reverting by week 24. Meanwhile, memory B cells showed a progressive increase over time (p = 0.01, Fig. 4A). These changes were more pronounced in antibody-producing vs non-antibody producing patients: for plasma cells / plasmablasts a difference in the increase of 13% at 12 week (p < 0.001) and for memory B cells an increase of 12.0% and 16.0% at 12 and 24 week (p = 0.006 and p < 0.001, respectively. Figure 4B). B cell response was not affected by disease severity ( Figure S8).
CD8 + T cell total numbers recovered progressively after week 6 (p < 0.001) but no changes were observed in CD4 + T cells (Fig. 4C). There was a reduction of naïve and effector CD8 + T cells and a progressive sustained increase in memory and TEMRA CD8 + T cells during the study period, regardless of disease severity (Fig. 4D and Figure S9). Of the transcription factors tested, only RORG gene expression levels were significantly high at week 6, progressively reducing thereafter (p = 0.001, Fig. 5A). Higher RORG expression was associated with poor outcome (persistent lung opacities (n = 2) and thrombosis (n = 3)) ( Figure S10 and Table S7). GATA3 levels showed a mild increase by week 12 (p = 0.022, Fig. 5A). All changes were more pronounced in antibody-producing vs non-producing patients: for RORG gene expression a decrease of 1.6 and 2.8 at 12 weeks and 24 weeks (p < 0.001) and for GATA3 gene expression an increase of 0.5 at 12 week (p = 0.005). In those with severe vs mild disease: for RORG gene expression an increase of 1.9 and 3.1 at 12 and 24 weeks (p < 0.001) and for GATA3 gene expression a www.nature.com/scientificreports/ or unexposed (Non-CoV2 +). *p < 0.05. (C) PBMCs from unexposed lupus patients with or without anti-SSA/Ro52 antibodies were stimulated with RBD protein, S1 protein or NS (non-stimulated condition, only PBS stimulation). After 24 h, PBMCs were analyzed by immunofluorescence to measure TRIM21 protein expression levels (green). Blue color (DAPI) labels cell nuclei. Error bars represent the mean ± SEM from three independent experiments. Scale bar = 50 µm. ***p < 0.0001. (D) TRIM21, IL4 and INFG gene expression was evaluated in PBMCs from unexposed SARS-CoV-2 lupus patients after RBD or S1 protein stimulation. Patients were subdivided into two groups according to the presence or absence of anti-SSA/Ro52 antibodies. In vitro stimulation was performed using RBD or S1 protein over 24 h. RT-qPCR gene expression analysis was performed thereafter. Fold change in expression level was calculated over non-stimulated conditions (PBS stimulation). **p < 0.005, ***p < 0.0001. All p-values were obtained using unpaired t-test. www.nature.com/scientificreports/ decrease of 0.6 at 12 weeks (p = 0.001, Fig. 5B). No significant changes were observed in Tbet and FOXP3 expression (Fig. 5A). Of the cytokines tested, only IFNG expression levels were significantly raised at weeks 6 and 12 reducing by week 24 (p < 0.001) regardless of antibody production or disease severity ( Fig. 5C and Figure S11).

Discussion
This study represents one of the largest cohorts of lupus patients from the early phases of the COVID-19 pandemic, in which we evaluate the risk of infection and characterize SARS-CoV-2-specific humoral and cellular immunity in PCR-confirmed COVID-19 patients. Several early studies sought to describe COVID-19 epidemiology in lupus. Using clinical registries, patients' surveys, and reviews of hospital records, studies also faced certain limitations like the exclusion of asymptomatic or mild cases, or PCR test accuracy and availability. Whereas many later studies showed that lupus patients were not at increased risk of COVID-19 6-10 , early small case studies suggested higher risk and worse outcomes [11][12][13][14][15][16] . We identified 29 lupus patients with RT-PCR-confirmed COVID-19 representing an elevated accumulated incidence of the disease compared with the general population (4.07% vs 1.55%).
In agreement with previous studies, we showed high doses of corticosteroids and immunosuppressive therapy to be risk factors for SARS-CoV2 infection and disease severity 30 . In our cohort other classic comorbidities described in the general population were not found to be predictive [31][32][33][34] . Our data supports the ineffectiveness of hydroxychloroquine to prevent COVID-19 disease 21,22 with similar rates of COVID-19 in hydroxychloroquinetreated and untreated patients. We have also identified HCWs and anti-SSA/Ro52 antibody positivity as risk factors for COVID-19 infection, but not disease severity. HCWs constitute a significant proportion of all COVID-19 lupus patients and represented 31% of all infections in our study. This is higher than the 4% to 29% range described in a meta-analysis, although severity and mortality in this group was low 35 . Data reflects the lack of adequate preparation and shortage of personal protective equipment at that time 35 . The other risk factor found was the presence of anti-SSA/Ro52 antibodies. Ro52/SSA, also known as TRIM21, is one of the major autoantigens recognized by the anti-Ro/SSA antibodies in lupus 36 . Patients with anti-SSA/Ro52 antibodies had lower TRIM21 expression compared with anti-SSA/Ro52 negative patients, and this reduction was more significant following COVID-19 infection. TRIM21, a cytosolic ubiquitin ligase, belongs to the tripartite motif-containing (TRIM) super family and has antiviral properties related to IFN signaling pathway regulation, the production of inflammatory mediators via NF-κB signaling, and by enhancing the Th1/Th17 response [37][38][39][40] . Knockdown of TRIM21 has been shown to impair innate immune response to viral infection 41 . Our in vitro studies showed that TRIM21 reduction decreased IL-4 and IFNγ cytokine expression levels. IL-4 and IFNγ induce cytotoxic T lymphocytes that provide an effective barrier to the establishment of viral infections 42,43 . By inhibiting TRIM21 and related cytokines, anti-SSA/Ro52 antibodies may impair the innate immune response in lupus patients contributing to a higher risk for COVID-19.
Severe disease occurred in 7 patients (24.13%), of whom 4 required ICU admissions and one died. Although limited by small numbers, these rates are higher compared to the ICU admission rates in the general population (13.79% vs 0.86%). Like previous studies 44,45 , our data did not show any association between COVID-19 severity and immunosuppressants or corticosteroids use. However, baseline hypocomplementemia, a classic feature of SLE and marker of disease activity, was a risk factor for severe COVID-19. The role of complement activation and its contribution to illness severity is being increasingly recognized [46][47][48][49] . The complement system is an arm of the innate immune response whose reactive cascade of cleavage products can coordinate the inflammatory antimicrobial response at sites of infection. However, complement activation is also associated with the excessive inflammatory response seen in patients with severe COVID-19, and the presence of a complement-mediated microvascular injury syndrome [50][51][52][53] . Proportion of positive or negative SARS-CoV-2 IgG antibodies in the 29 lupus patients with confirmed COVID-19 at 6, 12 and 24 weeks after onset of symptoms. Lupus patients were classified according their COVID-19 severity (mild and severe CoV2 +). (B) SARS-CoV-2 IgG titers in COVID-19 lupus samples according to disease severity (severe vs mild) at 6, 12 and 24 weeks after onset of symptoms. Optical density (O.D.) was measured at 450 nm. Data is represented as mean ± SEM. p value was obtained using paired t-test. ***p < 0.0001. (C) Proportion of CD19 + IgD − CD27 − (naïve B cells), CD19 + IgD + CD27 + (memory B cells) or CD19 + CD27 + CD38 + (plasma cells / plasmablasts) obtained by flow cytometry between lupus COVID-19 and non-COVID-19 patients (CoV2 + and non-CoV2 + ). : *p < 0.05, **p < 0.005, ***p < 0.0001. (D) RT-PCR COVID-19-confirmed lupus patients were classified according to presence or absence of SARS-CoV-2 IgG antibodies to RBD protein (anti-RBD IgG + or IgG − ) or by disease severity (severe or mild). Percentage of naïve, memory B cells and plasma cells/plasmablasts were analyzed at 12 weeks after onset of symptoms. **p < 0.005. (E) Representative flow cytometry plots of CD8 + total cells (CD3 + CD8 + ), CD8 + naïve cells (CD3 + CD8 + CD45RA + CCR7 + ), CD8 + memory cells (CD3 + CD8 + CD45RA − CCR7 + ),CD8 + effector cells (CD3 + CD8 + CD45RA − CCR7 − ) and TEMRA CD8 + cells (CD3 + CD8 + CD45RA − CCR7 − ) from COVID-19 confirmed patients vs. unexposed lupus patients. *p < 0.05, ***p < 0.0001. (F) Relative expression levels of transcription factors of Th1 (GATA3), Th2 (TBet), Th17 (RORG) and Treg (FOXP3) subsets in exposed and un-exposed lupus patients (CoV2 + and non-CoV2 + ). Gene expression was also studied according to SARS-CoV-2 antibody production (anti-RBD IgG + or IgG − ) and COVID-19 disease severity (severe or mild). Relative gene expression was calculated using the 2-ΔΔCt method and GADPH was used as endogenous control. *p < 0.05, **p < 0.005. (G) Interleukin production was measured by RT-qPCR gene expression analysis in exposed and unexposed lupus patients (CoV2 + and non-CoV2 + ). *p < 0.05, **p < 0.005. p-values are obtained using t-test in (C) to (G). www.nature.com/scientificreports/ Although the sample was small, no increased lupus activity in COVID-19 patients was observed. This may be due to better compliance with HCQ in view of early reports of its efficacy in COVID-19 prevention, the increment of corticosteroids received in severe cases and the Th2 skew observed during convalescence. Overall, whether SARS-CoV-2 can trigger SLE flares is still controversial [54][55][56] . For those studies showing a de novo SLE diagnosis or worsening disease following COVID19, caution needs to be taken since certain SLE clinical diagnostic criteria overlap with COVID-19 symptoms and autoantibodies can occur transiently in response to infections. In addition, it is difficult to evaluate SARS-CoV 2-related worsening SLE disease in a setting where patients suffered from psychological distress, faced difficulty in healthcare access and withdrew medication due to financial constraints or fear of being immunocompromised 57 .

Figure 4.
Longitudinal cellular profile revealed an enrichment of memory B and CD8 + T cell in exposed SARS-CoV-2 lupus patients. (A) Longitudinal study of the percentage of CD19 + IgD − CD27 − (naïve B cells), CD19 + IgD + CD27 + (memory B cells) or CD19 + CD27 + CD38 + (plasma cells / plasmablasts) at 6, 12 and 24 weeks after onset of symptoms in SARS-CoV-2 exposed lupus patients (N = 12). Significant differences were calculated in comparison with the 6-week timepoint using paired t-test. *p < 0.05, **p < 0.005, ***p < 0.0001. (B) Patients who produced SARS-CoV-2 antibodies against RBD protein (anti-RBD IgG + ) have significantly increased levels of memory B cells at Week 12 and 24, and antibody-producing cells (plasma cells / plasmablasts) at Week 12 compared with anti-RBD IgG − patients. No differences were found in naïve B cells. Fold change significant differences over time were calculated between groups by linear mixed models. *P < 0.05, ***p < 0.001. (C) By flow cytometry, CD3 + CD8 + T cell percentages were significantly increased after 6 weeks. No differences were found in CD3 + CD4 + T cells. p-values were obtained using paired t-test . *p < 0.05, **p < 0.005, ***p < 0.0001. (D) Longitudinal dynamics of CD8 + T cell subtypes in convalescent SARS-CoV-2 lupus patients at 6-, 12-and 24-weeks post symptom onset. The vertical axis shows the percentage of CD3 + CD8 + cells, and the horizontal axis shows time. Error bars represent the mean ± SEM from 12 lupus patients. Significant differences were calculated in comparison with the 6-week timepoint using paired t-test. **p < 0.005, ***p < 0.0001.  45 . Cohorts were similar except by the higher rates of Hispanic ethnicity in their study having a higher antibody production. Reported seroconversion rates after SARS-CoV-2 symptomatic infection range from 91 to 99% in the general population 58 . SARS-CoV-2 IgG antibodies are generally detected 2 weeks after infection and have a variable durability. As previously reported, most lupus patients (75%) with RT-PCR-confirmed SARS-CoV-2 infection developed SARS-CoV-2 IgG antibodies despite 69% being on immunosuppressants, and titers were correlated with disease severity [59][60][61] . Lack of antibody response was associated with prednisone doses ≥ 7.5 mg/day. The impact of corticosteroids on the humoral response is unclear. Whereas short courses have been associated with decrease serum of IgG and IgA concentrations, studies using pulses has not shown a detrimental effect 62,63 . We also found seronegative patients to receive more mycophenolate mofetil. MMF is known to suppress the humoral immune response to influenza vaccine in kidney transplant 64 and to mRNA COVID-19 vaccines in solid organ transplant recipients 65 . Although seroprevalence in healthy individuals during the same period 66,67 ranged from 4 to 20%, in our cohort it was much lower (0.78%). In the questionnaire, most patients reported that they implemented strict protective behaviors to avoid exposure.

Scientific Reports
Overall, the serological response was sustained, but antibody loss over serial measurements was observed in patients by week 24. They were patients with mild COVID-19 and receiving immunosuppression. Previous studies reported that SARS-CoV-2 specific antibodies disappeared in convalescent COVID-19 patients within 3 to 6 months, mainly in mild disease [68][69][70] . However, we did not find any significant immunological profile. SLE is characterized by an aberrant immune response and many immunopathogenic mechanisms overlap with those described in COVID-19. Due to restrictions in obtaining samples during the acute infection, we characterized only the cross-sectional and longitudinal SARS-CoV-2 cellular response in convalescent patients. While studies in the general population showed variable CD8 + T cell responses, we found a significant reduction of CD8 + T cell frequency compared to non-infected lupus patients that progressively recovered, and no differences in CD4 + T and NK cells 71 . Unlike the reported predominant Th1 response with undetectable antigen-specific Th2 or Th17 responses in general convalescent COVID-19 patients, we found a predominant Th17 with a mild Th2 response, more pronounced in severe disease and especially in those with pneumonia. Data on T-cell subtype changes is limited, with few longitudinal studies or studies evaluating patients with severe disease 72 . Th2 Figure 5. A decrease of Th17 signaling was observed in immune longitudinal analysis in exposed SARS-CoV-2 lupus patients. (A) Gene expression of transcription factors at 6-, 12-and 24-weeks post symptom onset (N = 12). The vertical axis shows the relative expression, and the horizontal axis shows time. Error bars represent the mean ± SEM from 12 lupus patients. Significant differences were calculated in comparison with the 6-week timepoint using paired t-test. *p < 0.05, ***p < 0.0001. (B) Gene expression of transcription factors for Th17 (RORG) and Th2 (GATA3) were evaluated longitudinally in convalescent SARS-CoV-2 lupus patients according to the production of antibodies against RBD protein (anti-RBD IgG + or IgG − ) and disease severity (mild or severe). Fold change significant differences over time were calculated between groups by linear mixed models *p < 0.005, ***p < 0.001. (C) Cytokines were evaluated longitudinally in convalescent lupus patients by RT-qPCR gene expression analysis. Significant differences were calculated in comparison to the 6-week timepoint. Only interferon gamma (INFG) was significantly decreased at 24 weeks using paired t-test. *p < 0.05. www.nature.com/scientificreports/ and Th17 responses have been found to be detrimental for recovery in severe COVID-19 72 , in part due to their association with lung injury 73 . The increase in Th17/Treg ratio may contribute to the uncontrolled release of pro-inflammatory cytokines and chemokines, which could result in aggravated inflammatory responses. While elevated Th17 cells have been detected in SLE 74 , in the absence of other observed lupus activity, this increment appears to be more strongly correlated to COVID-19 disease severity and poor outcome. Most patients with a persistent Th17 response developed lung fibrosis and thrombotic events during follow-up. A recent study has suggested a possible role of Th17 as a predictor of progression to critical disease in COVID-19 hospitalized patients 75 . Consequently the predominant Th17 response observed in our SLE patients could be related more for SARS-CoV-2 infection rather than SLE activity. Several reports have demonstrated that SARS-CoV-2 elicits a protective immune memory response after recovery 76,77 . We report B and T-cell responses in PBMCs from lupus patients 6 months post-infection despite immunosuppressive therapy. Overall, the development of B-cell memory to SARS-CoV-2 was significant and sustained. Notably, it has been reported that memory B cells targeting the Spike protein or RBD have been detected in most COVID-19 cases 78,79 . SARS-CoV-2-specific CD4 + and CD8 + T cells have been detected in 100% and ~ 70% of convalescent individuals a short time after resolution 79 . In severe COVID-19 cases, it has been reported that CD8 + T cells exhibit a high proportion of CD57 + terminal effector cells, together with significant decrease of naïve cell population leaving unresolved inflammation 6 months after infection 80 . Likewise, we found decreased CD8 + naïve T cells, increased TEMRA and CD8 + memory T cells during the study period regardless of COVID-19 severity.

Scientific
There are several limitations in our study. First, there are no data during COVID-19 infection or the first weeks after discharge for comparison. Second, the relatively small number of patients evaluated in some of the sub-studies did not allow us to draw strong conclusions. Further larger studies are needed to validate the findings reported here. Third, although antibody testing was mostly done as standard of care, there may be some degree of bias regarding which patients were willing to travel to the hospital. SLE patient could have strict protective behaviors to avoid COVID-19 infection exposure and it could also create a bias of the results. Fourth, we were not able to perform neutralizing antibodies assay due to laboratory biosafety limitations. Fifth, we did no measured antigen specificity for memory cells. Finally, some of the immune modifications may have been influenced by the treatment received in severe COVID-19 such as steroids and anti-IL6.

Conclusions
In summary, this study shows that SLE patients are at higher risk of SARS-CoV-2 infection compared to the reference population. We identified HCWs and anti-SSA/Ro52 antibody positivity as risk factors for infection and hypocomplementemia for disease severity. Our study also extends an understanding of the humoral and cellular immune response of SLE patients to COVID-19. Most patients developed a sustained humoral and memory immune response despite immunosuppressive therapy. Convalescent lupus patients had a predominant Th17 response in severe cases with poor outcome.